PROJECT SUMMARY: DTx technology leverages lipidation, the covalent conjugation of long-chain fatty acids (LCFA) to oligonucleotide therapeutics, to enable delivery of siRNA into cells and tissues outside of the hepatocyte. A lipid motif has been identified that enables the activity of siRNA in vivo, following intravitreal injection to the eye, and across multiple primary cells including neurons. DTx-lipidated siRNA is highly efficacious and at least an order of magnitude more potent at repressing mRNA expression when compared to conjugates of other fatty acids (e.g. DHA) that have been utilized to facilitate siRNA uptake into neurons in vivo or in vitro. In this application, DTx proposes to evaluate the potential of this novel technology to deliver siRNA and/or antisense (AS) to neurons in vivo to understand broadly, if the technology is useful for neurodegenerative diseases and more specifically, if it can be utilized to prevent neuronal degeneration in a rat model of retinitis pigmentosa (RP). The first aim, SA1, will explore whether DTx technology can enable the delivery of AS molecules into neurons as effectively as it enables siRNA. While applications of AS and siRNA technology to repress mRNA expression overlap to some degree, there are situations, due to their distinct modes of action, where utilizing one modality over the other can be highly advantageous. In this aim, an AS, previously demonstrated to have in vivo activity, will be conjugated to the DTx lipid motif. Its ability to repress mRNA expression both ex vivo in primary neurons and in vivo in retinal neurons will be evaluated through a combination of qPCR and quantitative in situ hybridization (q-ISH). SA2 will evaluate the potential of DTx-conjugated siRNA to repress mRNA expression following intracerebroventricular (ICV) injection to the brain. Its ability to repress mRNA expression in neurons will be evaluated by qPCR and q- ISH relative to DHA-conjugated siRNA, an approach demonstrated to enable siRNA activity following ICV injection to the mouse brain. The final aim, SA3, will evaluate whether DTx technology can be applied to siRNA to prevent neuronal degeneration in a rat model of RP driven by a mutation, P23H, in the rhodopsin gene. An siRNA that potently and selectively targets the P23H mutant allele will be conjugated to DTx technology and evaluated in vivo in the P23H rat RP model for the ability to selectively repress P23H expression to prevent photoreceptor cell death and to preserve photoreceptor function. In this application, we lay out a path to better understand our platform technology for neuronal indications in the eye and CNS. The long duration of action of siRNA/AS therapeutics, and the inherent safety advantages of keeping a drug confined to a single compartment, makes local delivery an attractive and commercially viable approach should we succeed in enabling siRNA and improving AS activity in the eye and brain. We expect data from the proposed studies to guide future grant phase 1 SBIR applications aimed at developing therapeutics for neurodegenerative diseases in the CNS such as Huntington?s, Alzheimer?s and Parkinson?s disease and provide the data and a compelling rationale for a phase 2 SBIR aimed at moving DTx-conjugated P23H AS/siRNA into early clinical development. !!